The therapeutic window for targeting CSC includes two fundamental properties, possibly involved in the failure of current therapies: the chemo resistance and the dysregulation of metabolism. These two features are probably connected, but the molecular mechanisms by which targeting metabolism could impair chemo resistance is not fully understood and deserves further investigations. Given the explosion of interest and information on cancer metabolism, it is hoped that new therapies will emerge in the next decades; nonetheless there are already some drugs approved for clinical use in certain types of cancer and others under preclinical examination.
The dependence of cancer cells on aerobic glycolysis (Warburg effect) for ATP generation has been exploited as a target for anticancer therapies and many possibilities have been proposed [59] (Table 4).
a) The reduction of glucose entry into the cell, even though the toxicity associated to the glucose deprivation may be a major challenge for the use of this approach. Nonetheless, there are some drugs that reduce glucose uptake. For example: Imatinib (Glivec) is a tyrosine kinase inhibitor that leads to the internalization of cell surface GLUT1 (glucose transporter) and reduce glucose uptake [60]; 2-deoxyglucose (2-DG), a glucose analog, competes with glucose for transport across the cell membrane and, inside the cells, it is phosphorylated by the early enzymes of the glycolysis into 2-DG-6-phosphate, thus inhibiting glycolysis [61].
b) The inhibition of some fundamental enzymes of glycolysis, such as PKM2. TT-232 is an inhibitor of this enzyme [62].
c) The inhibition of pentose phosphate pathways (PPP), deemed as an important pathway for its role in the synthesis of nucleic acids, that are indispensable for rapid cell proliferation, and for production of NADPH, that reduces the oxidative stress. An analog of NADP (6-aminonicotinamide, 6-AN), inhibits glucose-6 phosphate dehydrogenase (G6PD, the first enzyme of the PPP pathway), thus increasing oxidative stress and reducing tumor growth [63]. However, different results have been obtained by Herter and colleagues, who demonstrated the neurotoxicity of high doses of 6-AN and the lack of anticancer activity of well-tolerated dose [64].
d) The inhibition of HIF-1, one of the most important regulators of glucose metabolism in cancer cells. Topotecan is an inhibitor of HIF-1 and promotes oxidative phosphorylation in tumor cells [65].
e) The promotion of oxidative phosphorylation in cancer cells, dependent on glycolytic metabolism. Different mechanisms have been proposed for the activation of mithocondrial respiration, such as the down-regulation of lactate dehydrogenase (LDH) by small interfering ribonucleic acid (siRNA) to prevent the conversion of
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pyruvate in lactate [66] or the inhibition of pyruvate dehydrogenase kinase 1 (PDK1), which inhibits PDH the key enzymes of the TCA cycle, by antisense oligonucleotides [67].
Table 4: Anticancer therapies targeting aerobic glycolysis. Abbreviations: 2-DG, 2-deoxyglucose; LND, lonidamine; 3-BrPA, 3-bromopyruvate; 3-PO, 3-(3-pyridinyl)-l-(4-pyridinyl)-2-propen-l-one; 6-AN, 6- aminonicotinamide; DCA, dichloroacetate; GA, geldanamycin; 2ME2, 2-methoxyoestradiol; PX-478, S- 2-amino-3-[4′-N,N,-bis(2-chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride. Upadhyay M1, Samal J, Kandpal M, Singh OV, Vivekanandan P. The Warburg effect: insights from the past
decade. Pharmacol Ther. 2013 Mar;137(3):318-30.
Mechanism of action Target
gene Inhibitor(s) Reference(s)
Reduction of glucose entry into
the cell GLUT1
Imatinib Barnes et al., 2005
2-DG Maher et al., 2005
Phloretin Cao et al., 2007
Fasentin Wood et al., 2008
Inhibition of phosphorylation of
glucose HK
LND Floridi et al., 1981
3-BrPA Chen et al., 2007 and Zhang et
al., 2012
Mannoheptulose Board et al., 1995, Scatena et al.,
2008 and Pathania et al., 2009
Methyl jasmonate Madhok et al., 2011
Inhibition of fructose-6-PO4 to
fructose-1,6-bisphosphate PFK1 3-PO Clem et al., 2008
Inhibition of phosphoenolpyruvate to pyruvate PKM2 TT-232 Flourophosphates creatine phosphate oxalate, l- phospholactate Szokoloczi et al., 2005 Madhok et al., 2011 Suppression of pentose phosphate pathway
TKTL1 Oxythiamine Rais et al., 1999
G6PD 6-AN Kohler et al., 1970 and Varshney
et al., 2005. Promotion of pyruvate entry
into mitochondria PDK1 DCA Michelakis et al., 2008
Reduction of HIF-1 activity HIF-1α
Topotecan Rapisarda et al.,
2002 and Rapisarda et al., 2004
Digoxin Zhang et al., 2008
YC-1 Semenza, 2003
GA Isaacs et al., 2002
2ME2 Mabjeesh et al., 2003
PX-478 Welsh et al., 2004
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Moreover, recent studies suggest that targeting mitochondria is an attractive strategy for cancer therapy and the term “mitocans” has been used to classify mitochondria-targeted anticancer drugs [68]. The reason of this interest is that the Warburg’s hypothesis of the defects in mitochondria is not totally unfounded and cancer cells exhibit various degrees of mitochondrial dysfunction, such as changes in energy metabolism, increased transmembrane potential and ROS production. In particular, the increase in ROS production by cancer cells is associated with multiple changes in cellular functions, such as cell proliferation, migration, differentiation and apoptosis, all fundamental properties of cancer cells.
Finally, we need to mention the most innovative drugs used to target mitochondrial functions: Metformin and CPI-613.
Metformin (Figure 15), one of most widely prescribed oral hypoglycemic agents, has recently received increased attention because of its potential antitumorigenic effects that are thought to be independent of its hypoglycemic activity. This has been evaluated in multiple
in vitro and in vivo studies, and it is now being tested in clinical trials as an adjuvant to classic
chemotherapeutic regimens [69, 70]. Specifically, this compound exerts its pharmacological effects by inhibiting the mitochondrial ETC and by activating the AMPK pathway. Because, Metformin induces metabolic stress by reducing mitochondrial ATP production, it has been suggested that it could inhibit the growth of cancer cells by decreasing the cellular energy status.
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CPI-613 is an analog of lipoic acid capable of disrupting mitochondrial metabolism and it seems to exhibit selective effects against cancer cells both in vitro and in vivo [71]. CPI-613 is an innovative drug, actually in clinical trial, targeting two key enzymes of the TCA cycle: pyruvate dehydrogenase kinase (PDK) and alpha-ketoglutarate dehydrogenase (α-KGDH) (Figure 16). In specificity, it stimulates PDK and consequently it induces the inactivation of PDH through phosphorylation, activating multiple tumor cell death pathways. Moreover, it induces of α-KGDH with an endogenous redox mechanism. So this single drug simultaneously and independently attacks two central, essential mitochondrial metabolic complexes.
Figure 16: CPI-613 and target therapy in mitochondria. Paul M. Bingham and Zuzana Zachar The Pyruvate
Dehydrogenase Complex in Cancer: Implications for the Transformed State and Cancer Chemotherapy.
The possibility that Metformin could efficiently target CSC [69, 70], suggests that this rare cell population is characterized by a different metabolism that the cancer cells, privileging a mitochondrial-dependent metabolism.
In view of the novelty of the drug, there is no significant evidence about the capacity of CPI- 613 to induce the death of CSC. It has to keep into account that CSC could have different metabolic profiles depending on their tissue of origin and their degree of differentiation. For example, highly undifferentiated liver cancers tend to be more glycolytic than differentiated
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cells [73], while a recent study using glioma stem cells show that these cells consume less glucose and produce less lactate, compared to their cancer cell counterpart [74].
In conclusion, the idea that CSC are characterized by a different metabolism than the cancer cells, could open new avenues to new target therapies.
The niche is the source of molecules that activate or inhibit signal transduction pathways and, while the stem cell microenvironment of a normal tissue is known to maintain a balance between self-renewal and differentiation, the tumor microenvironment predominantly displays proliferating signal to cancer cells and CSC. Accordingly, anti-metabolic reprograming strategies begin to provide a roadmap for the generation of novel “metabo- stemotoxic” therapies, counteracting therapeutic resistance, cancer aggressiveness and
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1.7 Aims of the project
The aims of this project are:
a) The phenotypic characterization of CSC in ascitic effusions from ovarian cancer- bearing patients. The characterization will be carried out through the application of techniques such as: stemness marker expression, spheroid formation, chemo resistance assay and tumorigenicity in vivo.
b) The investigation of the metabolic profile of CSC isolated from patients with epithelial ovarian cancer, through specific metabolic assays to evaluate whether the CSC fraction is characterized by the known Warburg effect or by other particular metabolic pathways.
These studies have the ambition to define differences between CSC population and the non- stem counterpart, in order to discover specific therapeutic targets, because it is known that the niche protect CSC from drugs and specific nutrient or oxygen stress.
For this reason, we will also focus our attention on a key nutrient stress, glucose deprivation, to test the addiction of CSC to this nutrient and their ability to survive under starvation conditions. These experiments could in fact demonstrate in vitro the ability of CSC to persist in stress condition.
The final aim is to find a specific drug or drug combination that targets the relevant metabolic pathways.